Bioluminescence resonance energy transfer (BRET) occurs naturally in marine organisms such as Aequorea victoria and Renilla reniformis (Morin and Hastings, 1971). BRET is a form of Förster resonance energy transfer (RET), which is the non-radiative transfer of energy from an excited state donor to a ground state acceptor. There are two commonly used forms of the BRET principle, i.e., BRET1 and BRET2. Both use Renilla luciferase (RLuc) as the energy donor. In BRET1, the substrate is native coelenterazine (CLZ) or coelenterazine h (CLZh). RLuc and a yellow fluorescent protein (YFP) are the energy donor and acceptor, respectively, giving peak donor emission at 475 nm and peak acceptor emission at 535 nm. In BRET2, YFP is replaced with GFP2 and a modified CLZ substrate, i.e. coelenterazine-400a or (CLZ400a) is used. The peak donor emission and acceptor emission are shifted to 395 nm and 515 nm, respectively (Dacres et al., 2009a, b; Pfleger and Eidne, 2006). A third form of BRET, i.e., BRET3, has recently been developed. It used CLZh as the substrate and RLuc8 as the energy donor and mOrange as the acceptor, resulting in improved spectral resolution (De et al., 2009).
RET is a ratiometric technique which can eliminate data variability caused by fluctuations in light output due to variations in assay volume, assay conditions and signal decay across different wells in a plate. RET-based reactions are homogeneous, generally occurring in solution without solid-phase attachment. This allows for detection of analytes in different forms such as liquid, gas and even particulates without separation. The avoidance of solid-phase attachment eliminates the process of surface regeneration used in many surface-based techniques such as Surface Plasmon Resonance (SPR) (Fang et al., 2005) and, in conjunction with the fast reaction rate, allows it to be used for on-line monitoring.
So far, however, uses of BRET have been restricted to research laboratories using sophisticated detection equipment. Microfluidic technologies are attracting interest in many fields, including chemistry, biology, medicine, sensing and materials. Their advantages over conventional technologies include reduced reagent consumption, fast reaction rate, short analysis time, and amenability to automation and mass production (Holden and Cremer, 2005).
There have been substantial research and development in microfluidic technologies. Examples include an integrated biochip design with fluorescence light collection (EP 2221606), on-chip biosensing using Raman spectroscopy (WO 2009/020479), a biosensing device (WO 2009/018467) for detecting GPCR-ligand binding using surface plasmon resonance techniques, a light detection chip (US 2011/0037077 and US 2008085552) with mirrors as light reflectors, an assay device with a cartridge format (WO 2009/044088), a chemiluminescence-based microfluidic biochip (US 2002/0123059) and so on. Many of these device have the disadvantages of high cost per chip due to integration of multiple components, inability to perform real-time monitoring due to the requirement for surface regeneration and slow reaction of reagents, limited detection sensitivity, or signal drift.
Furthermore, there is considerable background art in the fields of electronic noses and electronic tongues, which contact a gaseous or liquid sample with an array of solid state sensors in order to detect analytes and/or classify the samples. Electronic noses and tongues have been bedevilled by poor performance due to limited selectivity of the sensors, poor sensitivity, sensor saturation and slow regeneration and sensor drift over time.
There is therefore a need for further methods of detecting analytes and classifying samples based on the analytes they contain, particularly methods that can be performed in real time and with increased sensitivity and that do not suffer from downtime due to the need to regenerate the sensing surface and that resist the confounding effects of sensor drift. Multiple channel microfluidic systems deploying an array of biologically derived sensors electro-optically coupled to a detection system offer a novel solution to these problems.